INTRODUCTION - Mammalian L1 elements (long interspersed repeated DNA elements, also called LINE-1) belong to the non-LTR class of retrotransposons. An L1 element replicates (retrotransposes) by copying its RNA transcript into genomic DNA. The ~6 kb human L1 element has four regions: the 5?-untranslated region (UTR) has a regulatory function; open reading frame 1 (ORF1) encodes an RNA binding protein; ORF2 encodes an endonuclease and reverse transcriptase; the 3?-UTR contains a conserved G-rich polypurine motif & ends in an A-rich tail. L1 elements replicate by using the 3? OH of the nicked target DNA to prime the synthesis of a cDNA copy of its transcript. L1 can also replicate other elements (e.g., SINEs such as the Alu family) by this process. As L1 activity has persisted in mammals since their emergence ~100 million years ago and the products of L1 replication have been largely retained, it is not surprising that at least 30% (860 Mb) of the human genome was generated by L1 activity: 460 Mb is L1 DNA and 360 Mb is SINE DNA. In addition to their sheer mass, L1 elements, and their SINE offspring, can cause genetic rearrangements and inactivate or alter gene activity. We have used the tools of molecular biology and evolution and population genetics to examine the interaction between L1 and its host and how L1 activity might affect modern humans. RECENT FINDINGS: ANALYSIS OF AN ONGOING L1 AMPLIFICATION EVENT IN HUMANS ? We have now published our studies using an unbiased method to collect Ta1 L1 element inserts from four ethnic groups: African pygmy, Caucasian Druze, Chinese, Melanesian. We showed several years ago that Ta1 is the most recently emerged & most active L1 family in humans. Our cloning strategy recovered 90% of the Ta1 inserts in the four individuals; 40% of which are not present in the human genome database, and of these 93% are polymorphic as compared to 51% for the database entries. Thus, the database seriously under represents both the Ta1 census in humans and its contribution to human genetic diversity. Ta1 insertions are not random with respect to GC content but have an insertional bias toward GC poor regions. Ta1 elements are also not randomly distributed between or within chromosomes as chromosome 4 contains twice the number expected for its length and gene density. In contrast, ancestral families did not insert preferentially on chromosome 4. Furthermore, the distribution of Ta1 elements within individual chromosomes is not uniform. Ta1 elements tend to cluster, and the size of the maximal gaps between Ta1 inserts is larger than would be expected by chance. Neither the clustering nor chromosomal bias can be explained on the basis of GC content. In a separate study we determined the distribution of 120 of the polymorphic Ta1 inserts in 141 individuals and are now analyzing these data. Also we began a collaboration with Lynn Jorde and Mark Batzer to determine the distribution of our polymorphic inserts in 1500 individuals. EVOLUTIONARY DYNAMICS OF L1 IN NON-HUMAN PRIMATES ? We have published our investigation of L1 evolution in three species of New-World monkeys (NWM): the squirrel monkey (Saimiri sciureus), the tamarin (Saguinus oedipus) and the spider monkey (Ateles paniscus). Since these three species diverged, L1 has amplified in the Saimiri and Saguinus lineages but L1 activity seems to have been strongly reduced in the Ateles lineage. In addition, the active L1 lineage has evolved rapidly in Saimiri and Saguinus generating species-specific subfamilies. In contrast, we found no evidence for a species-specific subfamily in Ateles, a result consistent with the low L1 activity in this species for the last ~25 My. We also found that two L1 lineages coexisted in the common ancestor of these three species for at least 6 million years, but only one of them has persisted until the present time. The coexistence of more than one L1 lineage for such a long tiiiiime is very unusual in mammals where a single dominant lineage is the rule. One possible explanation for the existence of a single L1 lineage is competition between active L1 elements for a limiting host factor(s) essential for L1 replication. As the competition would presumably be reduced during periods of low L1 activity, this latter condition could favor the co-existence of multiple active L1 lineages. In fact, multiple L1 lineages seem to typify non-mammalian species (e.g., Drosophila, fish) that do not support the high level of L1 activity (or other non-LTR retrotransposon activity) that seems to be the case most of the time in mammals. INTERACTION BETWEEN L1 AND ITS HOST - We had earlier found that the coiled coil motif of L1 ORF 1 had undergone episodes of adaptive evolution early in hominid evolution and that this ceased during the evolution of L1 in the African apes (human, chimpanzee, gorilla). As coiled coil domains can mediate protein-protein interaction, evolutionary change in this motif could reflect interaction of L1 with host factors. Using the tools of molecular evolution as a guide, we reconstructed the ancestral coiled coil domain and created a fully ancestral coiled coil domain and two versions of partially ancestral domains in the context of an otherwise modern element. All but one of the partially ancestral ORF1 is active for retrotransposition. This suggests that the adaptive evolution was not a response to changes elsewhere in the L1 element but possibly to the host. We used the cytoplasmic yeast two-hybrid assay to identify clones in various expression libraries (e.g., human fibroblasts (HeLa cells) & testes) that encode factors that may interact with ORF1. So far we have isolated 30 different such host proteins and as we had hoped some that interact with the modern coiled coil do not interact with the ancestral coiled coil. Clearly then evolutionary change in the host has accompanied the evolution of L1. In other experiments we found that coiled coil is absolutely essential for L1 retrotransposition and we have begun to determine just what amino acids changes in the coiled coil render it inactive for retrotransposition. We have also begun to examine the interaction of L1 with the host protein, nuclear exchange factor (NXF) 1. NXF1 mediates nuclear export of non-spliced RNAs, as would be the case for L1 RNA and some retroviral RNAs. As L1 elements in mammals totally dominate the retrotransposon landscape, this could result from L1 pre-empting NXF1. And finally we have completed phase 1 of a rapid retrotransposition assay to examine the effect of putative host factors on this process.